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Page 1: Cure for thalassemia major – from allogeneic …llogeneic hematopoietic stem cell transplantation has been well established for several decades as gene replacement therapy for patients

214 haematologica | 2017; 102(2)

Received: March 20, 2016.

Accepted: October 12, 2016.

Pre-published: December 1, 2016.

©2017 Ferrata Storti Foundation

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Ferrata StortiFoundation

EUROPEANHEMATOLOGYASSOCIATION

Haematologica 2017Volume 102(2):214-223

REVIEW ARTICLE

doi:10.3324/haematol.2015.141200

Allogeneic hematopoietic stem cell transplantation has been wellestablished for several decades as gene replacement therapy forpatients with thalassemia major, and now offers very high rates

of cure for patients who have access to this therapy. Outcomes haveimproved tremendously over the last decade, even in high-risk patients.The limited data available suggests that the long-term outcome is alsoexcellent, with a >90% survival rate, but for the best results, hematopoi-etic stem cell transplantation should be offered early, before any endorgan damage occurs. However, access to this therapy is limited in morethan half the patients by the lack of suitable donors. Inadequatehematopoietic stem cell transplantation services and the high cost oftherapy are other reasons for this limited access, particularly in thoseparts of the world which have a high prevalence of this condition. As aresult, fewer than 10% of eligible patients are actually able to avail ofthis therapy. Other options for curative therapies are therefore needed.Recently, gene correction of autologous hematopoietic stem cells hasbeen successfully established using lentiviral vectors, and several clinicaltrials have been initiated. A gene editing approach to correct the b-glo-bin mutation or disrupt the BCL11A gene to increase fetal hemoglobinproduction has also been reported, and is expected to be introduced inclinical trials soon. Curative possibilities for the major hemoglobin dis-orders are expanding. Providing access to these therapies around theworld will remain a challenge.

Cure for thalassemia major – from allogeneichematopoietic stem cell transplantation togene therapyAlok Srivastava1*and Ramachandran V. Shaji1

1Department of Haematology & Centre for Stem Cell Research (a unit of inStem,Bengaluru), Christian Medical College, Vellore- 632004, Tamil Nadu, India

ABSTRACT

Introduction

Thalassemias are the most common human monogenic disorders related to thedeficiency of the production of either the α- or b-globin chains.1 b-thalassemia is alarger clinical problem because its homozygous form, thalassemia major, leads tosevere morbidity and mortality due to very low endogenous hemoglobin levelswhich are incompatible with life.2 Even though educating the society at large, massscreening, cohort counselling and prenatal diagnosis can be applied to very effec-tively reduce the incidence of b-thalassemia major, this has only been achieved insome countries.3 More than 50,000 children with this disease are born worldwideeach year, adding to the disease burden of this condition.4 Hypertransfusion andiron chelation have been the mainstay of therapy for thalassemia major for nearly50 years.5 However, these therapies are often ineffective due to complications relat-ed to repeated transfusions and inadequate iron chelation. The main reasons for thisare the lack of compliance with the planned therapy due to its logistic demands aswell as the ongoing costs of chelation therapy. This approach, therefore, leads tosignificant morbidity and mortality with up to 50% of these patients developingsignificant organ dysfunction by the time they are adults, even in Western coun-tries.6 This figure is even higher for patients in developing countries.7 The need forcurative therapy for thalassemia major was addressed with the success of allogeneichematopoietic stem cell transplantation (alloHSCT), which was initiated in theearly 1980s as a way to replace the defective gene in such patients.8 AlloHSCT

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remains the only widely available curative therapy for thiscondition at present. The best results are seen whenalloHSCT is offered early, before complications related toiron overload or transfusion-transmitted infections set in,with survival rates of over 90% being reported in thesepatients.9The outcomes of higher risk patients have also continu-

ously improved over the last two decades, so that 80-90%long-term survival rates have been obtained even in thisgroup of patients.10,11 However, there are many challengesin offering alloHSCT as a therapy for these patients allover the world.12 More recently, gene replacement inautologous hematopoietic stem cells (autoHSCs) usingviral vectors has become a reality, witGST M1 null sta-tus'h several clinical trials showing its success and poten-tial for wider use.13 This article will address the status ofalloHSCT for b-thalassemia major and also briefly reviewthe newer options for gene correction in autoHSCs usingdifferent approaches.

Allogeneic Hematopoietic Stem CellTransplantationWhile the classical principles of alloHSCT remain the

same in treating patients with thalassemia major, earlyresults showed that there were special challenges relatedto pre-existing organ dysfunction, particularly involvingthe liver as well as the hyperactive immune system, prob-ably related to repeated transfusions.14 Given that most ofthese patients were children with a non-malignant dis-ease, busulfan (14-16mg/kg total dose) and cyclophos-phamide (160-200 mg/kg total dose) based conditioningwas chosen, even though many of them had liver dysfunc-tion related to iron overload or transfusion-related viralhepatitis.8 Significant regimen-related toxicities (RRT)were observed if the doses of the drugs were intensified,while there was a high incidence of graft rejections if thedoses were reduced. In high-risk patients, this led to mor-tality rates of up to 35% and rejection in nearly 30% ofpatients, resulting in long-term event-free survival of onlyabout 50%.15 High RRT was attributed to the sequentialuse of these two drugs, often with severe sinusoidalobstruction syndrome, particularly in those with signifi-cant liver dysfunction.16 RRTs were also shown to be asso-ciated with certain genetic polymorphisms in the glu-tathione S transferase M1 (GSTM1) and the cytochromeP450 (CYP450) genes. Detailed pharmacogenetic studiesshowed that the GST M1 null status and the CYP2C9gene polymorphisms affected the pharmacokinetics ofbusulfan and cyclophosphamide, with a possible impacton RRTs.17,18 Therefore, modifications in the approach toalloHSCT for these patients were needed.Over the last decade, the results of alloHSCT have

improved significantly. This has been possible due to bet-ter risk stratification, more effective targeted dose adjust-ment of intravenous busulfan during conditioning, a mod-ified conditioning regimen and continually improving sup-portive care.19,20 Patients over 7 years of age withhepatomegaly of more than 5cm have been identified asan especially high-risk group who are candidatesfor/require novel approaches, particularly with regard totheir conditioning regimen and preparation for trans-plant.21,22Two approaches have been taken in this regard, one

based on pre-transplant immunosuppression using flu-darabine, and the other by introducing a longer gap

between the use of busulfan and cyclophosphamide dur-ing conditioning23,24 or using less toxic myeloablativeagents such as treosulfan and avoiding cyclophosphamidecompletely.11 All these modifications have led to signifi-cantly improved survival rates of nearly 80-90% in thesehigh-risk patients (Table 1).10,11,15,19,20,23-27While we have to appreciate the impact of successful

alloHSCT on the lives of these patients, we need to con-tinue to recognize the many challenges that persist withrespect to the still significant morbidity and mortalityassociated with this procedure. All over the world a majorconstraint is the lack of access to this therapy related tothe lack of a suitable donor. Matched related donors aregenerally available only for a third of patients with tha-lassemia major,28 but in countries with larger families orcommunities with consanguineous marriages up to two-thirds of patients may have suitable related donors.29However, only a very small minority of the global patientpopulation falls into this category. The need for alternativedonors has therefore been explored in several ways - par-tially mismatched related donors,30 related haploidenticaltransplants,31,32 and matched unrelated donors, the latter ofwhich could be adult humans or cryopreserved cord bloodunits in blood banks.20,33 While good results have beenachieved with alternative donors in some studies, experi-ence is still limited and the outcomes variable. Thereforethese approaches have not yet been translated intobecoming the standard of care for thalassemia major.28Persisting concerns regarding high rejection rates and graftversus host disease (GvHD) need to be addressed throughthe evaluation of novel protocols in future studies.If these challenges can be addressed, haploidentical

alloHSCTs could become a major alternative to matchedunrelated donors or cord blood transplants. This could beparticularly advantageous in resource-constrained coun-tries with large numbers of these patients, where access tounrelated donors is severely restricted due to lack of serv-ices and the associated costs.32-34The aim of curative therapy for any disease is to eradi-

cate it and also enable a normal life afterwards. With highsurvival rates in patients with thalassemia major undergo-ing alloHSCT, the next important issue relates to the man-agement of pre-existing and late transplant-related compli-cations and their impact on long-term survival.6,15 Over90% of patients who survive the first two years afteralloHSCT are generally expected to become long-term sur-vivors.35 Late (more than 2 years) complications afterHSCT for hematological malignancies and bone marrowfailure syndromes are accounted for mostly by diseaserelapses, chronic GvHD, infections and second cancers.36In addition, particularly in children, growth retardation,multiple endocrine and other organ dysfunction and cog-nitive changes have also been noted, depending on theirpre-existing co-morbidities at the time of alloHSCT, or ifthey had developed major post-transplant complicationssuch as significant chronic GvHD. Since most of thealloHSCTs for b-thalassemia major involve children andadolescents, data on long-term outcomes becomes partic-ularly important as they would be expected to have sever-al decades of life post-HSCT. This is even more relevant inpatients receiving repeated transfusions because many ofthem already have systemic complications related tochronic anemia, iron overload and resultant endocrine andmetabolic dysfunction, in addition to transfusion-trans-mitted infections.37 Organ dysfunction exists in almost

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75% of patients undergoing alloHSCT for thalassemiamajor with endocrine, hepatic and cardiovascular diseaseaccounting for nearly 30-40% each, along with a host ofother organ dysfunctions.34 Unfortunately, information onlate complications in ex-thalassemic patients is very limit-ed. The only very long-term (over 20 years) study of out-come after alloHSCT for thalassemia major has shownthat patients who were well-managed before transplantcan have a quality of life comparable to the normal popu-lation, and certainly better than those being managed con-servatively with hypertransfusion and iron chelationalone.38 Older age at alloHSCT and the development ofsignificant chronic GvHD were found to be factors leadingto a worse long-term outcome. More data with a systematic evaluation of organ dys-

function as well as an analysis of possible pre-transplantcontributing factors is therefore needed in ex-thalassemicpatients. An important issue will be the extent of ironoverload at HSCT and the effectiveness of iron chelationpost-HSCT, as this is the major cause of organ dysfunctionin these patients. This is another aspect of the long-termoutcome in these patients which has not been adequatelyaddressed.39,40 Varied approaches to iron chelation havebeen reported in small numbers of patients afteralloHSCT, ranging from phlebotomies if serum ferritinwas >2000 ng/ml,39 to using only iron chelators such asdesferrioxamine at 40mg/kg or deferasirox at

20-30mg/kg.41,42 The time at which chelation was initiatedhas also varied from shortly after engraftment to up totwo years after HSCT. A more considered and coordinat-ed approach is needed to initiate intensive iron depletiontherapy as soon as possible after alloHSCT in order toreduce or stop continuing organ damage. As the vast majority of these patients come from the

Mediterranean, Middle-Eastern and Asia-Pacific regions,where there is poor access to optimal transfusion-chela-tion therapy pre-HSCT, particular attention should be paidto iron depletion therapy in the post-HSCT period,7,40 asmany patients develop significant organ dysfunction at anearly age, often within the first decade. A clear strategyneeds to be implemented for improving pre-HSCT man-agement with hypertransfusion and iron chelation, select-ing patients early for HSCT before any major organ dys-function sets in, and for the post-HSCT management ofthese residual complications. This is particularly impor-tant for all those patients who were poorly chelated beforeHSCT, and are closer to puberty. Without such anapproach, while better survival may be achieved even inolder higher risk patients, it is unlikely that these patientswill have a normal quality of life because of the pre-exist-ing irreversible morbidities. In fact, they will end uprequiring multidisciplinary management of those dysfunc-tions post-HSCT. An additional feature after HSCT for major hemoglobin

Table 1. Major reported clinical studies that have attempted to improve the outcome of patients with class 3 thalassemia major$. Year N Median Proportion Proportion Major defining feature of change in protocol Treatment Graft EFS OS

Age (yrs) / in Class in Class related rejection (%)(range) 3 (%) 3HR (%) mortality (%) (%)

Lucarelli et al.15* 1996 115 11 (3-16) 100 NA Bu / Cy based regimen with reduction in Cy total dose 24 35 49 74from 200 mg/kg to 160 mg/kg

Sodani et al.25 2004 33 11 (5-16) 100 NA Reduction in Cy dose to ≤160 mg/kg with addition of Flu. 6 6 85 93Additional therapy from day -45 with immunosuppression with Azt and suppression of erythropoiesis with HU

Gaziev et al.19 2010 71 9 (1.6–27) 57.3 NA Intravenous Bu, dose adjustments with 7 5 87 91therapeutic drug monitoring

Chiesa et al.26 2010 53 8 (1-17) 47 NA Intravenous Bu, dose adjustments with 4 15 79 96therapeutic drug monitoring

Chiesa et al.26# 2010 25 NA 100 NA Intravenous Bu, dose adjustments with 4 34 66 96therapeutic drug monitoring

Bernardo et al.10 2012 60 7 (1-37) 27^ NA Treo based conditioning regimen 7 9 84 93Li et al.20 2012 82 6 (0.5-15) NA NA Conditioning with age adjusted PK based IV Bu, 8.5^ 4^ 88^ 91^

Cy (110mg/kg), high-dose Flu (200mg/kg), Thio. Additional therapy from day -45 with immunosuppression with Azt and suppression of erythropoiesis with HU

Choudhary et al.27 2013 28 9.6 (2-18) 75 39 Treo based conditioning regimen 21 7 71 79Anurathapan et al.23 2013 18 14 (10-18) 100 NA Conditioning regimen of Flu &IV Bu Pre-conditioning 5 0 89 89

immunosuppression therapy with Flu and Dexa for 1-2 months.

Mathews et al.11 2013 50 11 (2-21) 100 48 Treo based conditioning regimen with PBSC graft in 74% 12 8 79 87Mathews et al.11@ 2013@ 24 12 (3-21) 100 100 Treo based conditioning regimen with PBSC graft in 74% 13 8 78 87Gaziev et al.24 2016 37 10 (5-17) 100 NA As in Sodani et al.25 but with higher dose 8 0 92 92

of Flu (150 mg/kg) and addition of Thio(10 mg/kg)$Adapted from Mathews et al.10; *Only patients <17 years included in this table; #Subset of high-risk cases from same paper; ^Includes all adult cases as well (assumed to be Class 3); ^Includes low-risk patients also; @Subset of high-risk cases from same paper. Cy: Cyclophosphamide; Flu: Fludarabine; Dexa: Dexamethasone; Bu: Busulfan; Treo: Treosulfan; Azt: Azathioprine; HU: Hydroxyurea; Thio:Thiotepa. HR: high-risk; EFS: event-free survival; OS: overall survival; NA: not applicable; PK: pharmacokinetics; PBSC: peripheral blood stem cell.

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disorders is the state of mixed chimerism. This refers tothe persistence of significant levels of residual host cells(RHC) of the hematopoietic system post-HSCT classifiedas level 1 (<10%), level 2 (10-25%) and level 3 (>25%).Transient mixed chimerism (TMC) is not uncommon inthe immediate post-HSCT period and can occur in 30-45% of patients in the first 100 days.43 Depending onthe level of RHC, patients with TMC may later becomecomplete chimeras in 60-75% of cases for those at level 1,but in only 5-30% of cases of those with level 3. (Figure 1)However, these predictions are based on limited data.Therefore careful monitoring of TMC and correlationwith the hemoglobin levels and other blood counts is nec-essary post-HSCT in patients with major hemoglobin dis-orders. It should also be noted that some patients develop

persistent mixed chimerism (PMC), in which a significantcomponent of RHC persist long-term without rejection ofthe graft.44 In some patients, the RHC component could bemuch higher than 25% with an acceptable hemoglobinlevel. If stable, then it is obviously a case of PMC of differ-ent hematopoietic elements and immune tolerance in sucha way that it does not affect the erythroid lineage adverse-ly. T regulatory cells could play a role, but the exact mech-anism of this phenomenon is not fully understood.45 Theassessment of subset chimerism, particularly of the ery-throid and lymphoid lineage in more patients, as shown inFigure 2, could shed more light on the mechanismsinvolved.46 While RHC levels below 50% in T cells hasbeen reported to be associated with a very low risk ofrejection, high levels of RHC in both T and NK cells are

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Figure 1. Evolution of chimerism after hematopoietic stem cell transplantation (HSCT). Early mixed chimerism is associated with higher risk of rejection while latechimerism often persists with a stable graft. RHCs: residual host cells. MC: mixed chimerism.

A

B

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associated with rejections in up to 90% of patients.47These parameters also need further evaluation.More HSCTs are now being done in the Asia-Pacific

region for hemoglobin disorders than in any other part ofthe world,48 given the high need and greater support fromhealth care authorities as well as an increasing number ofcenters offering these services. It is therefore of the utmostimportance and necessity to better assess the elementsthat will give the best long-term outcomes. This requiresthe complete evaluation and documentation of non-hema-tological complications pre-HSCT as well as the late post-transplant complications, in order that they can be appro-priately managed to allow these ex-thalassemic patients tolead as normal a life as possible.38 To this end, attentionshould be especially directed towards educating thesefamilies and the health care providers early about thepotential complications and their prevention, as well asthe importance of safe and effective transfusion-chelationprograms. This will help keep patients in the best condi-tion possible for alloHSCT to be undertaken. Indeed, ourgoal should be to offer alloHSCT to all patients with majorhemoglobin disorders well before they become high-riskdue to end-organ damage. As we persist with our efforts to increase access to

alloHSCT and its outcome, it is very important to note itslimitations from a public health perspective. While morethan 50,000 children with b-thalassemia major are addedto the world population every year, less than 5,000alloHSCTs have been reported so far for this conditionworldwide over the last 30 years.28 Even accounting forunder-reporting, the actual number is likely to be wellbelow 10,000 during this period. With the majority ofthese patients being in the Mediterranean, Middle-Eastern

and Asia-Pacific regions of the world, access to this thera-py remains highly restricted due to the lack of centerswith trained personnel and facilities for HSCT services,and the inability of patients to access care due to the highcosts, which very often have to be met by the patients ortheir families.7,49 Recent data from the Asia Pacific Bloodand Marrow Transplantation Registry shows that onlyabout 450 alloHSCTs are being reported from the regionevery year, and perhaps less than 1,000 worldwide (per-sonal communication, lida M, APBMT Registry) The enor-mity of this mismatch means that a different strategy isneeded for effectively managing the large numbers of suchpatients in the world. Two approaches are necessary toaddress this problem. First, a preventative strategythrough effective population screening, genetic coun-selling and prenatal diagnosis should be initiated in allcountries where the prevalence of these conditions arehigh.3 Second, the development of a more practical cura-tive therapy, both in terms of technology, logistics andcost, if possible, such as gene correction in autoHSCs.50This will perhaps be the best way by which large numbersof patients who cannot avail of alloHSCT for various rea-sons will be able to access a curative therapy without theinherent risks and potential complications of alloHSCT.

Gene therapy for b-thalassemia major

Much effort has been put into developing gene therapyfor several monogenic hematological disorders over thelast several decades.51 For those disorders where the defectis due to mutations in single genes that affect hemoglobinsynthesis, the approach has been to introduce a normal

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Figure 2. Methods to detect red cell chimerism. In patients with late persistent mixed chimerism, erythroid precursors may be predominantly donor while the non-erythroid cells may be more recipient in origin. Red cell chimerism can be measured by analyzing donor specific RBC antigens, STRs in erythroid DNA or nucleotidevariations in erythroid transcripts. BFU-E: burst forming unit – erythroid; CFU-E: colony forming unit – erythroid; STR: short tandem repeats; RBC: red blood cell.

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gene into autoHSCs and, more recently, to actually repairsuch genes with various genome editing tools.52,53 The ini-tial success achieved with the retroviral vectors used forefficient gene transfer into autoHSCs for different forms ofsevere combined immunodeficiency (SCID) disorders53was tempered by the development of clonal diseases andovert leukemia.54 These vectors had intact long terminalrepeats (LTRs) which included efficient and ubiquitousenhancers. While this aided the desirable high expressionof the target transgene, it also led to undesirable genotox-icity.53,54 The activation of oncogenes such as LMO2 in theSCID trial led to the development of acute lymphoblasticleukemia in some of these patients. However, soon there-after, the development of lentiviral vectors devoid of theirpathogenic elements and with a self-inactivating (SIN)design provided a better alternative, not only in terms ofits safety but also its ability to infect quiescent HSCs andcarry larger transgene cassettes, which are needed formore effective expression in order to produce gram quan-tities of hemoglobin.55 These advances in HSC-based genetherapy laid the foundation for its application in the majorhemoglobin disorders. The first successful gene therapy for a major b-globin

disorder was in 2007 when a patient with bE/b0-tha-lassemia was treated.56 The process involved the harvest-ing of autologous HSCs from the recipient, followed by

ex vivo transduction of these cells with the lentiviral vectorcarrying the transgene. (Figure 3) If these transduced HSCswere found suitable following quality assessment to deter-mine the number transduced as well as the vector copynumbers per HSC, they were used to perform an autolo-gous HSCT after appropriate conditioning therapy todestroy existing HSCs. With a transduction efficiency ofabout 30% and only 10-20% of HSCs showing the trans-gene expression, and a busulfan-based myeloablative con-ditioning regimen in the initial patient, there was only lim-ited expression of b-globin in the first year after gene ther-apy. This patient, therefore, required several transfusionsduring this time. In the second year, however, the trans-gene expression gradually improved and led to about 2-3g/dL of transgene-associated HbA, resulting in the overallhemoglobin stabilizing at 8.5 to 9.0 g/dL and the patientbecoming transfusion-independent.57 However, this wasalso associated with a clonal expansion of erythroid cells(10-12%), with the insertion site being the high mobilitygroup AT-hook 2 (HMGA2) locus. This clone peaked at4% of hematopoietic cells at about 4 years, but has sincedeclined to about 1% at 5 years without a reduction intotal hemoglobin.58 More recent data from this group hasshown that with further improvements in the lentiviralvector leading to greater transduction efficiency and high-er vector copy numbers, more than 15 patients have now

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Figure 3. Overview of current approaches to gene therapy for the major hemoglobin disorders. Gene modifications may be through viral vectors or genome editingtechnologies to achieve the desired therapeutic effect. HSC: Hematopoietic stem cell; BM: Bone marrow; PB: Peripheral blood; ZFN: zinc finger nucleases; TALEN:transcription activator-like effectors with Fokl nuclease; CRISPR: clustered regularly interspaced short palindromic repeats.

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been treated.59,60 Most of these patients achieved muchhigher transgene expression resulting in the hemoglobinincreasing by 4-6 g/dl within the 2-5 months followinggene therapy.58,61 Significantly, no clonal events have beendescribed thus far in these patients. The level of transgene-associated hemoglobin expression is related not only tothe quality of transgene construct, but also to the efficien-cy of transduction itself with any vector, which in turncould be dependent on certain intrinsic properties of theparticular HSC.62 While much more needs to be learntabout effective gene therapy for major hemoglobin disor-ders, these results have certainly set the stage for furtherclinical trials to be undertaken. Several more studies arenow open for patient recruitment.63-66 Initial results contin-ue to be encouraging and there have not been any signifi-cant safety concerns so far.While this method of gene therapy for the major hemo-

globin disorders relies on substituting a functional b-globin gene for a defective one, another approach couldbe to increase fetal hemoglobin (HbF) production byswitching on the γ-globin gene expression which becomesgradually suppressed in the months following birth.67 Amajor regulator of this phenomenon has been shown to bethe BCL11A transcription factor which could itself be dis-rupted to reinitiate γ-globin gene expression through cell-intrinsic mechanisms.68 This could then combine with thenormal α−globin to produce enough HbF in these patientsto effectively correct their anemia.69 This approach hasindeed been shown to be effective in animal models andcultured human HSC derived erythroid cells, where inac-tivation of the BCL11A gene increased HbF production toreverse sickle cell disease.70,71 This has also now found clin-ical applications as described below.There are many challenges ahead related to several

aspects of gene therapy for the major hemoglobin disor-

ders. Not least of these is the vector design, which is ofutmost importance in order to ensure efficient transduc-tion in HSCs to produce clinically meaningful high expres-sion of the required globin chain that can then result innear-normalization of the hemoglobin in these patients.The success of these challenges needs to be achievedwithout dangerous genetic perturbations from theunavoidable random integration of the lentiviral vectors,which can lead to clonal hematopoietic disturbances withtheir own serious implications. Another aspect that canrestrict success is the survival advantage of the transducedHSCs, given the fact that even with myeloablative condi-tioning, untransduced HSCs will also find their way backinto the hematopoietic compartment in the patient, giventhe variable ex vivo transduction efficiency of the lentiviralvectors. Unlike alloHSCT, there is no donor immune sys-tem in this form of autologous stem cell therapy to give asurvival advantage to the transduced transplanted HSCswith the functional b-globin gene. The dynamics that willdetermine the sustained presence of the transduced HSCsin the long-term are still unknown.

Genome editing for gene correction

Another approach to correct mutations in autoHSCs isthrough genome editing techniques using targeted nucle-ases, which can specifically target these sites and replacethem with the normal sequence to bring back the wild-type functional configuration. These technologies includethe zinc fingers nucleases (ZFN) and the transcription acti-vator-like effector nucleases (TALENS) and more recently,the clustered regularly interspaced short palindromicrepeats (CRISPR) with Cas9 nuclease system.52Conceptually, these powerful technologies allow for a

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Figure 4. Genome editing techniques for correction of molecular defects. The three different gene editing techniques allow fixing of single mutations in the hemo-globin gene in patients with b-thalassemia major. ZFNs: zinc finger nucleases; TALENs: transcription activator-like effectors with Fokl nuclease; CRISPR: clusteredregularly interspaced short palindromic repeats.'

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D

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molecular ‘cut and paste’ approach to gene correction(Figure 4),72 which could transform the entire approach togene therapy for many diseases. In fact, the speed withwhich these applications are entering the clinic is remark-able.73 The ability to efficiently and safely edit the genometo modify genes has indeed taken both therapeutics anddiagnostics by storm. It has also given a new dimension tothe potential of stem cell-based therapies to treat differentdiseases. A clinical trial has already been reported showingthe safety and limited efficacy of the ZFN based system inaltering the CCR5 receptor on lymphocytes from patientswith HIV infection on highly active antiretroviral therapy(HAART) therapy.74 Thus far however, the major chal-lenge in using gene editing strategies in the correction ofb−thalassemia is the low efficiency of these methods intargeted gene correction in HSCs. This is necessary forobtaining a large number of gene corrected HSCs to pro-duce therapeutically significant levels of hemoglobin.However, a recent study that used improved methods todeliver ZFNs, showed that integrase-deficient lentiviralvectors to deliver ZFN messenger ribonucleic acids(mRNAs) and an oligonucleotide as a gene correction tem-plate had a very high efficiency of gene correction in HSCsobtained from patients with sickle cell disease.75,76 A simi-lar approach can be applied in b-thalassemia major.The correction of b-globin gene mutations in induced

pluripotent stem cells (iPSCs) derived from patients withthalassemia major is also possible.77-81 One advantage ofusing iPSCs for gene correction is that it could then be pos-sible to obtain a completely corrected clone of pluripotentstem cells, from which a large number of stem cells orother cells of interest could be derived for transplantation.However, so far it has not been possible to use iPSC-derived HSCs to engraft and provide sustainedhematopoiesis.82 On the other hand, an encouraging proofof concept comes from the report of using ZFN technolo-gy to correct the interleukin 2 receptor (IL2R) gene in dis-eased human HSCs and progenitors, and the demonstra-tion in animal models of their ability to sustain adequatehematopoiesis after transplantation.83 In fact, SangamoBioSciences, Richmond, CA, USA, has recently obtainedapproval from the U.S. Food and Drug Administration(FDA) for the Investigational New Drug11 application forSB-BCLmR-HSPC for the treatment of b-thalassemia,

developed in collaboration with Biogen, Cambridge, MA,USA.84 Data presented more recently by this groupclaimed about 70% efficiency of gene knockout using thisapproach in HSCs manipulated ex vivo.85 Clearly, these are very significant developments that

have created more options for gene replacement or correc-tion therapies for b-thalassemia major and other serioushemoglobin disorders (Figure 3). It may then be possibleto move from alloHSCs as vehicles for the transfer of thenormal gene to viral vectors or genome editing techniquesbeing used to correct the gene defect in autoHSCs. Onecannot underestimate the enormity of the work ahead inestablishing the safety and efficacy of the latter approach-es, but given the nature of the technology, it is not difficultto imagine that they hold great promise for providing plat-forms that could be much more amenable to developingcost-effective therapies for applications across the globe.At least they will not be limited by the much more diffi-cult task of finding suitable donors for alloHSCT and thesignificant treatment-related immediate and long-termcomplications associated with it. However, these are veryearly days with regards to gene therapy, and many moreclinical trials and follow-up studies will be needed toestablish the long-term safety of the different approachesto gene therapy for the major hemoglobin disorders. As with any technology-based options to therapeutics,

intellectual property rights based restrictions on the useof effective technologies along with the cost and market-ing policy related issues can lead to hugely restrictedaccess to such therapies around the world.86 Solutionswill need to be found for those challenges if such situa-tions do arise. Even with all these unresolved issues,there is no doubt that these are very exciting times forpatients, physicians and scientists working in this fieldbecause after years of great effort, not only have theresults of alloHSCT improved significantly for thesepatients, but several innovative gene correction therapiesare also on the horizon.

AcknowledgmentsThe authors would like to like to thank Mr. Tamil Vanan J.

from the Centre for Stem Cell Research, Christian MedicalCollege Campus, Bagayam, Vellore, India for his assistance withthe preparation of this manuscript.

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haematologica | 2017; 102(2) 221

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